ABSTRACT The plasticity and functional diversity of regulatory T cells (Tregs) is essential for the maintenance of healthy immunity but it is also exploited in disease. We have described a subset of Tregs that express high levels of ?- catenin and promote inflammation. The frequency of these Tregs is increased in inflammatory bowel diseases (and mouse colitis), colon cancer (and mouse polyposis), as well as in multiple sclerosis (and mouse EAE). Expression of a dominant active ?-catenin, or ablation of Tcf-1 in Tregs both foster expression of Ror?t the canonical transcription factor of the Th17 lineage, and promote a Th17 differentiation signature. In the mesenteric lymph nodes (MLN) of healthy mice, we identified at least 5 transcriptionally distinct activated clusters of Tregs. One of these clusters is defined by expression of Ror?t and has prominent Th17 differentiation characteristics. In Tcf-1 deficient Tregs the Th17 differentiation profile expanded to all other clusters, and the ROR?t cluster increased in frequency. The Tcf-1 deficient Tregs in spite of being more Th17 like, suppressed T-cells, however they failed to suppress inflammation. This indicates a bifurcation of Treg suppressive functions and begs the question, of whether it involves a co-operation between TCF-1 and Foxp3. We have found that, TCF-1 and Foxp3 co-bind enhancer elements of genes involved in T cell activation and Th17 differentiation. ?-catenin induces newly accessible chromatin regions at these enhancers and upregulates the expression of the associated genes. Moreover, the E-box binding protein HEB works together with TCF-1 and potentially also Foxp3 to regulate these genes, since Treg specific ablation of both TCF-1 and HEB (but not each one alone) rescues inflammatory and autoimmune pathologies associated with ?-catenin activation in Tregs. Consistently, HEB regulates ROR?t, represses Foxp3 and peripheral Treg development. Our findings are in line with the notion that FoxP3 directly activates or represses transcription, in a context- and partner-dependent manner. They further implicate ?-catenin, TCF-1, and HEB in partnering with Foxp3 to independently regulate the diverse Treg suppressive activities. Based on these findings we hypothesize that, Wnt/?-catenin signaling differentially regulates Treg suppressive functions and diversity, by controlling access of Foxp3 and HEB to select chromatin sites bound by Tcf-1. To address this hypothesis in specific Aim 1 we will determine the role of ?-catenin and Tcf-1 in preparing the epigenetic landscape for Foxp3 binding and in shaping Treg cell type diversity. In specific Aim 2 we will determine the role secreted Wnt ligands in defining the functional properties of colon infiltrating Tregs. The proposed studies will elucidate fundamental co- operations/antagonisms between TFs that regulate Treg properties in health, and how microenvironment queues like Wnts exploit them in disease settings. The expected findings have the potential to inform novel diagnostic and therapeutic tools for autoimmunity and cancer.